Date of Award

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Research Advisor

Peter J. McKinnon, Ph.D.

Committee

Lawrence M. Pfeffer, Ph.D. Susan E. Senogles, Ph.D. Richard J. Smeyne, Ph.D. Jian Zuo, Ph.D.

Keywords

ATM, ATR, brain, DNA damage, DNA repair, MRN complex

Abstract

DNA double strand breaks create a situation of extreme stress under which a cell must either be capable of repairing the lesions in order to continue replication or succumb to death. Not surprisingly, deficiencies in DNA repair genes often lead to human diseases frequently associated with genomic instability, cancer proneness, and neuropathology. Neurological consequences of aberrant DNA repair mechanisms vary depending upon the affected gene and the pathway in which it operates. Ataxia-telangiectasia (A-T) is the prototypical disease associated with DNA double strand break (DSB) repair deficiency and is characterized by severe neural pathology. A-T results from homozygous mutations that inactivate the ataxia-telangiectasia mutated (ATM) gene, and typically presents in early childhood as cerebellar ataxia accompanied by ocular telangiectasias, elevated α-fetoprotein levels, and lymphoma predisposition compounded by radiosensitivity. The ATM protein is a large (~380 kDa) protein kinase within the PI3K family, which mediates cell cycle checkpoint activity and induces apoptosis through phosphorylation of copious substrates. It is thought to be through apoptotic induction of newly post-mitotic cells harboring lethal levels of DNA damage that Atm maintains integrity of the CNS, as it prevents incorporation of genomically compromised neurons into the mature nervous system. Hypomorphic mutations in the Mre11/Rad50/NBS1 complex (MRN complex), which is required for efficient ATM activity, result in distinct syndromes. Ataxia-telangiectasia-like disease (ATLD) results from truncating mutations in Mre11 and presents with neurodegeneration similar to that observed in A-T, but with longer latency and less severity. Mutations in the FHA and BRCT domains of NBS1 result in Nijmegen Breakage Syndrome (NBS), which is characterized by microcephaly without evidence of neurodegeneration. Thus far mutations in Rad50 have not been reported in humans. In order to better understand the roles of ATM and MRN signaling in maintaining physical and functional integrity of the CNS, mutant mice either null for Atm or carrying hypomorphic mutations in Mre11 or Nbs1 were subjected to either endogenous or exogenous forms of DNA damage and the signaling response was assessed in the brain. To understand the contrasting neuropathology resulting from Mre11 or Nbs1 hypomorphic mutations, we analyzed neural tissue from Mre11ATLD1/ATLD1 and Nbs1DB/DB mice after genotoxic stress. We found a pronounced resistance to DNA damage-induced apoptosis after ionizing radiation or DNA ligase IV (Lig4) loss in the Mre11ATLD1/ATLD1 nervous system that was associated with defective Atm activation and phosphorylation of its substrate p53. Apoptosis occurred normally in the Nbs1ΔB/ΔB brain. We also conditionally disrupted Lig4 throughout the nervous system using Nestin-cre (Lig4Nes-Cre), and while viable, these mice showed pronounced microcephaly and a prominent age-related accumulation of DNA damage throughout the brain. Either Atm-/- or Mre11ATLD1/ATLD1 genetic backgrounds, but not Nbs1ΔB/ΔB rescued Lig4Nes-Cre microcephaly. Thus, DNA damage signaling in the nervous system is different between ATLD and NBS, and likely explains their respective neuropathology. Ataxia-telangiectasia and Rad3-related (ATR) is a large serine/threonine kinase that is highly similar in structure to ATM and phosphorylates many overlapping substrates. Hypomorphic mutations in ATR have been associated with some cases of Seckel syndrome, a microcephalic condition. Despite the similarities between ATM and ATR, inactivation of either kinase has quite distinct consequences. Unlike ATM, ATR is essential for embryonic development, as Atr-/- embryos are lethal by E7.5. To better understand the differing roles of ATM and ATR in the nervous system, a conditional Atr mutant was crossed onto the Nestin-cre transgenic line. AtrloxP/loxP; Nestin-cre ( AtrNes-cre) animals displayed early post-natal lethality and severe neurogenesis defects, particularly striking in the cerebellum due to decreased proliferation and viability of granule neuron progenitors. Surprisingly, progenitors in the rhombic lip were largely unaffected in AtrNes-cre embryos whereas the granule cell precursors of the cerebellar external germinal layer (EGL) displayed DNA damage as early as E13.5 and p53-dependant apoptosis by E15.5. Between E15.5 and E16.5 p53 independent cell cycle arrest was inferred by decreased proliferation of the remaining granule cell precursors in the EGL. DNA damage was not observed in the rhombic lip until E16.5. The forebrain showed a similar pattern of acquired damage followed by apoptosis and decreased proliferation. The p53-null genetic background did not substantially rescue the AtrNes-cre phenotype despite blocking apoptosis. This data indicates that proliferation arrest is the major factor causing ATR-Seckel syndrome and that ATR does not significantly contribute to p53-induced apoptosis in proliferating neural progenitors. AtrNes-cre animals were crossed onto an Atm conditional background (AtmNes-cre) in order to assess how ATM and ATR cooperate in maintaining homeostasis in the brain. The AtmNes-cre background failed to alter the AtrNes-cre phenotype, underscoring the divergence of ATR and ATM function during embryogenesis, despite overlapping substrates between the kinases. In summary, this work provides us with important insight into the requirements for specific DNA damage signaling molecules in the nervous system. ATM and ATR appear to have distinct roles in the nervous system with ATM required for elimination of compromised early post-mitotic neurons, while ATR is critical for maintaining integrity and replicative capacity of progenitor populations. Based upon this study, there is no obvious cross-talk or overlap between Atm and Atr signaling in the nervous system as each plays a separate role in different cell populations. Furthermore, we have begun to tease out the existence of independent signaling roles between Mre11 and Nbs1 in regard to Atm activity. While Mre11 mutations associated with ATLD compromise Atm-dependant apoptosis, Nbs1 hypomorphism linked to NBS does not appear to negatively impact upon known Atm function in the nervous system. Overall these data provide new insights into the specific genetic requirements for DNA double strand break repair in the nervous system.

DOI

10.21007/etd.cghs.2009.0290

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